This project involves the development of continuous cell lines, for use as models to investigate effects of drugs in vitro, in neural transplantation as an alternative to primary cells and tissues, and for basic studies of neural cell biology. Current efforts include development of mutant truncated forms of SV40 large T antigen and methods for the delivery of genes or combinations of genes to cells in order to modify the cell cycle. A mutant form of SV40 large T antigen, which lacks p53 binding activity, has been cloned to examine those properties of SV40 large T antigen which are required for immortalizing CNS neurons. This mutant oncogene, called T155, is capable of overcoming cell cycle arrest and immortalizing primary mesencephalic neurons. T155 appears to interfere with the expression of differentiated phenotypes to a much smaller degree than wild-type SV40 large T antigen, and, moreover avoid the problem of interference with the activity of p53 which could promote the survival of cells with DNA damage. Primary mesencephalic cell cultures immortalized with T155 express differentiated neuronal markers (e.g., neurofilament) and glial markers (e.g., GFAP), whereas cells immortalized with wild-type SV40 large T rarely express markers characteristic of mature neurons or glia. A further series of variants of the T155 oncogene have been developed in order to obtain the minimal oncogene required for immortalization of neuronal precursor cells. These oncogene fragments are being employed in order to obtain immortalized cell lines. In one set of experiments, striatal cell lines have been obtained that produce high levels of GABA, and these cells are effective in transplantation studies in animals. Cell lines from kidney epithelium that produce high levels of growth factors and can be used in neural transplantation in animal models of stroke have also been produced. Human progenitor cell cultures have been expanded and have been shown to be capable of differentiating into mature neurons and astrocytes. Clonal populations of these cells have been immortalized with telomerase and T155, in order to develop lines with homogeneous properties, that can consistently differentiate into specific neuronal phenotypes. These studies may lead to the production of human cell lines that could be used for therapeutic purposes and as in vitro models for studying effects of drugs of abuse.